Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase.
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ABSTRACT:
SUBMITTER: Knorrscheidt A
PROVIDER: S-EPMC8496131 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the kcat/Km value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.
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Project description:Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme's activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.
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Project description:Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae, so that neutral mutations were simply added and recombined in vivo With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombination in vivo IMPORTANCE Fungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift and in vivo DNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed in S. cerevisiae.
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Project description:A continuous flow approach for the aerobic photo-oxidation of benzylic substrates to ketone and aldehyde products is presented. The resulting process exploits UV-A LEDs (375 nm) in combination with a Corning AFR reactor that ensures effective gas-liquid mixing and leads to short residence times of 1 min. A variety of benzylic substrates are converted to their corresponding carbonyl products, and scalability is demonstrated to produce multigram quantities of products within a few hours. Overall, this continuous flow approach offers several improvements over alternative oxidation methods due to the combined use of air as an oxidant and SAS (sodium anthraquinone-2 sulfonate) as a water-soluble photocatalyst. The use of greener and safer conditions together with process intensification principles renders this flow approach attractive for further industrial applications.
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Project description:Lipid mediators, such as epoxidized or hydroxylated eicosanoids (EETs, HETEs) of arachidonic acid (AA), are important signaling molecules and play diverse roles at different physiological and pathophysiological levels. The EETs and HETEs formed by the cytochrome P450 enzymes are still not fully explored, but show interesting anti-inflammatory properties, which make them attractive as potential therapeutic target or even as therapeutic agents. Conventional methods of chemical synthesis require several steps and complex separation techniques and lead only to low yields. Using the newly discovered unspecific peroxygenase TanUPO from the ascomycetous fungus Truncatella angustata, 90% regioselective conversion of AA to 14,15-EET could be achieved. Selective conversion of AA to 18-HETE, 19-HETE as well as to 11,12-EET and 14,15-EET was also demonstrated with known peroxygenases, i.e., AaeUPO, CraUPO, MroUPO, MweUPO and CglUPO. The metabolites were confirmed by HPLC-ELSD, MS1 and MS2 spectrometry as well as by comparing their analytical data with authentic standards. Protein structure simulations of TanUPO provided insights into its substrate access channel and give an explanation for the selective oxyfunctionalization of AA. The present study expands the scope of UPOs as they can now be used for selective syntheses of AA metabolites that serve as reference material for diagnostics, for structure-function elucidation as well as for therapeutic and pharmacological purposes.
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Project description:Unspecific peroxygenases (UPO) are glycosylated fungal enzymes that can selectively oxidize C-H bonds. UPOs employ hydrogen peroxide as oxygen donor and reductant. With such an easy-to-handle co-substrate and without the need of a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from Hypoxylon sp. EC38 (HspUPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris. The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. HspUPO is a versatile catalyst performing various reactions, such as the oxidation of prim- and sec-alcohols, epoxidations and hydroxylations. Semi-preparative biotransformations were demonstrated for the non-enantioselective oxidation of racemic 1-phenylethanol rac -1b (TON = 13000), giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated yield. HspUPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with small-to-medium size substrates of variable polarities. The structural investigation of several protein-ligand complexes revealed that the active site of HspUPO is accessible to molecules of varying bulkiness and polarity with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, HspUPO is an exploitable candidate for peroxygenase-based biocatalysis.
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